Methods and materials for producing recombinant viruses in eukaryotic microalgae

Abstract

The present invention is directed to methods and materials for producing recombinant viruses. In particular, methods and materials are provided for producing recombinant viruses in eukaryotic microalgae such as Chlamydomonas reinhardtii. Recombinant adeno-associated viruses are examples of recombinant viruses produced according to the invention.

I claim: 
     
       1. A eukaryotic microalgae that produces a recombinant adeno-associated virus (rAAV), wherein the rAAV is a viral vaccine vector or a viral gene therapy vector, and the genome of the rAAV comprises inverted terminal repeats flanking a vaccine or gene therapy polynucleotide. 
     
     
       2. The eukaryotic microalgae of  claim 1  wherein the eukaryotic microalgae is  Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella sorokiniana, Chlorella kessleri, Volvox carteri, Dunaliella salina, Ostreococcus tauri, Phaeodactylum tico, Gonium pectoral , and  Cyanidiosschyzon merolae.    
     
     
       3. The eukaryotic microalgae of  claim 1  wherein the eukaryotic microalgae is  Chlamydomonas reinhardtii.    
     
     
       4. The eukaryotic microalgae of  claim 1 , wherein the microalgae is transformed with a polynucleotide construct comprising (i) a polynucleotide sequence encoding Rep 78 protein, Rep 52 protein, VP1 protein or_VP2/3 protein or (ii) one or more polynucleotide sequences encoding Rep78 protein, Rep52 protein, VP1 protein and VP2/VP3 protein or (iii) one or more polynucleotide sequences encoding Rep78 protein, Rep52 protein, VP1 protein, VP2 protein and VP3 protein. 
     
     
       5. The eukaryotic microalgae of  claim 1 , wherein the microalgae is transformed with one or more polynucleotide constructs wherein each polynucleotide construct comprises at least one polynucleotide sequence encoding Rep 78 protein, Rep 52 protein, VP1 protein, VP2/3 protein, VP2 protein or VP3 protein. 
     
     
       6. The eukaryotic microalgae of  claim 5  wherein the construct further comprises an AAV inverted terminal repeat (ITR) and 3′ AAV ITR, and helper functions for generating a productive AAV infection. 
     
     
       7. The eukaryotic microalgae of  claim 1, 2, 3, 4, 5 or 6 , wherein the rAAV is recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13. 
     
     
       8. The eukaryotic microalgae of  claim 1 , wherein the rAAV is recombinant AAV9. 
     
     
       9. The eukaryotic microalgae of  claim 1 , wherein the vaccine or gene therapy polynucleotide is operatively linked to promoter DNA and a polyadenylation signal sequence. 
     
     
       10. The eukaryotic microalgae of  claim 1 , wherein the vector genome further comprises an intron sequence. 
     
     
       11. A method of producing a recombinant adeno-associated virus (rAAV), wherein the rAAV is a viral vaccine vector or a viral gene therapy vector, comprising the steps of growing a eukaryotic microalgae producing the rAAV,
 wherein the genome of the rAAV comprises inverted terminal repeats flanking a vaccine or gene therapy polynucleotide. 
 
     
     
       12. The method of  claim 11  wherein the eukaryotic microalgae is transformed with a polynucleotide sequence expressing the recombinant virus. 
     
     
       13. The method of  claim 11 or 12  further comprising the step of purifying the rAAV. 
     
     
       14. The method of  claim 11 or 12 , wherein the eukaryotic microalgae is  Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella sorokiniana, Chlorella kessleri, Volvox carteri, Dunaliella salina, Ostreococcus tauri, Phaeodactylum tico, Gonium pectoral , and  Cyanidiosschyzon merolae.    
     
     
       15. The method of  claim 11 or 12 , wherein the eukaryotic microalgae is  Chlamydomonas reinhardtii.    
     
     
       16. The method of  claim 11 , wherein the eukaryotic microalgae is transformed with a polynucleotide construct comprising (i) a polynucleotide nucleotide sequence encoding Rep 78 protein, Rep 52 protein, VP1 protein or VP2/3 protein or (ii) one or more polynucleotide sequences encoding Rep78 protein, Rep52 protein, VP1 protein and VP2/VP3 protein or (iii) one or more polynucleotide sequences encoding Rep78 protein, Rep52 protein, VP1 protein, VP2 protein and VP3 protein. 
     
     
       17. The method of  claim 11 , wherein the eukaryotic microalgae is transformed with one or more polynucleotide constructs wherein each construct comprises at least one polynucleotide sequence encoding Rep 78 protein, Rep 52 protein, VP1 protein, VP2/3 protein, VP2 protein or VP3 protein. 
     
     
       18. The method of  claim 16 or 17  wherein the polynucleotide construct further comprises an AAV inverted terminal repeat (ITR) and 3′ AAV ITR, and helper functions for generating a productive AAV infection. 
     
     
       19. The method of  claim 11 , wherein the rAAV is recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13. 
     
     
       20. The method of  claim 11 , wherein the rAAV is recombinant AAV9. 
     
     
       21. The method of  claim 11 , wherein the vaccine or gene therapy polynucleotide is operatively linked to promoter DNA and a polyadenylation signal sequence. 
     
     
       22. The method of  claim 11 , wherein the vector genome further comprises an intron sequence.